Most anti-cancer drugs target binding pockets in enzymes or on protein receptors. These drugs often target upstream pathways that can cripple tumors, but may fail to hit the central node of the tumor. For some time, it has been known that transcription factors can serve as these central nodes. Indeed, de-regulated transcription has been associated with most, if not all, properties that are critical to the cancer cell, including uncontrolled growth, unlimited replicative potential, migration and invasion. Yet few investigators have taken on the more difficult task of targeting transcription factors. The Six1 homeobox gene encodes a transcription factor that is critical for embryonic development. In most tissues, Six1 expression is lost once development is complete. However, Six1 is re-activated in as many as 50% of primary breast tumors and 90% of metastatic lesions, as well as in many other tumor types. Six1 plays a causal role in tumor initiation, tumor growth, and metastasis of breast and other cancers, and its inhibition dramatically diminishes the aforementioned properties in several mouse models of cancer. Six1 has no intrinsic activation or repression domains, and thus requires cofactors to mediate its transcriptional effects. We have demonstrated that Six1 requires the Eya cofactor to mediate its pro-proliferative and pro-metastatic effects in breast cancer cells. Examination of public breast cancer microarray datasets reveals that high expression levels of Six1 and Eya together, but not either gene alone, correlate with shortened time to relapse, shortened time to metastasis, and shortened overall survival in breast cancer. Importantly, we recently determined the crystal structure of the Six1/Eya2 complex, which reveals that Six1 uses a single alpha helix to interact with Eya, resembling two well-known protein- protein interactions (p53/HDM2 and Bak/Bcl-xL) that have been successfully targeted by small molecules. We further demonstrated that a single amino acid mutation on the Six1 helix abrogates the binding of Six1 to Eya and Six1-mediated tumor initiation and metastasis. The goal of this project is to identify and develop novel anti- cancer agents that target the Six1/Eya complex through characterizing and optimizing hit compounds identified through the use of a high throughput screen (Aim 1). Identified inhibitors will be tested in both cell culture and animal models for their ability to inhibit tumorigenicity and metastasis (Aim 2). Since the Six1/Eya complex influences multiple stages of the tumorigenic and metastatic processes and is not expressed in most adult tissues, targeting this never before targeted complex has the therapeutic potential to inhibit breast and other cancers both at early and later stages of disease progression with limited side effects, something most current therapies do not do.